Project Summary The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase. Our research has made major contributions to provide structures of RNA polymerase at the different stage of transcription cycle. During the next five years, we will continue to study the transcription machinery in bacteria and archaea to provide insights of the fundamental mechanism of transcription, which is conserved from bacteria to human. Over the last 20 years, X-ray crystallography has been playing a major role in the structural biology of RNA polymerase and revealed many important structures. RNA polymerases at evident stages in the mainstream of the transcription cycle are often referred to RNA polymerase core enzyme, holoenzymes, open complex with a strong promoter, transcription initiation and elongation complexes. These structures have been well characterized due to their stable natures. A challenge in the structural biology of cellular RNA polymerase is to visualize transient interactions of RNA polymerase with other regulatory factors and ligands that occur in between each evident stage in the mainstream or at the branched pathways from the mainstream of transcription cycle. Due to their elusive natures, crystallization of such macromolecular assemblies has been a bottleneck and thus limited the approach by X-ray crystallography. In the last couple of years, the resolution of macromolecular structures determined by cryo-electron microscopy (cryo-EM) has been drastically improved due to multiple technical advances, allowing us to visualize heterogenous and large assembly of macromolecular complexes. We will use structural biology methods including both cryo-EM and X-ray crystallography together with other biochemical approaches to visualize these transient interactions and investigate their effects for transcription process. Major targets of our study are: 1) the interaction between the Escherichia coli RNA polymerase and DksA/ppGpp for transcription regulation during the stringent response; 2) the interaction between bacterial RNA polymerase and ATP-dependent motor enzyme for rescuing stalled RNA polymerase at the end of transcription cycle; and 3) the interaction between archaeal RNA polymerase and ATP-dependent transcription termination factor Eta for disrupting a stalled transcription elongation complex to initiate the transcription-coupled DNA repair.